HCV dependencies on the host machinery are both intricate and extensive. Each of these host dependencies is a potential therapeutic target. Previous efforts have been successful in discovering important steps in HCV replication, yet many fundamental processes in the viral life cycle remain uncharacterized. Using RNAi-based genetics and an infectious HCV cell culture system, we performed an unbiased genome-wide screen to identify host factors required for productive HCV infection. Among the identified host factors, we studied E-cadherin (CDH1), an adherens junction protein, as a novel HCV-specific entry factor. CDH1 depletion by siRNA drastically reduced HCV entry and productive infection in Huh7.5.1, HepG2/miR122/CD81 cells and primary human hepatocytes. Silencing of CDH1 also inhibited HCV infection in a single-cycle infection assay, reinforcing the importance of the gene at an early step of HCV life cycle. CDH1 siRNA had no effect on HCV replicon or IRES assays. Anti-CDH1 monoclonal antibody efficiently blocked the infection of both HCV pseudoviruses (HCVpp) and HCVcc in a dose-dependent manner. We showed that CDH1 depletion had no effect on the expression levels of four major HCV entry factors CD81, SR-BI, claudin-1 (CLDN1) and occludin (OCLN). By confocal microscopy, CDH1 appeared to closely associate with CLDN1 and OCLN on the cell surface. Silencing of CDH1 drastically reduced the cell surface distribution of CLDN1 and OCLN, providing a mechanism whereby CDH1 is involved in HCV entry. CDH1 thus plays a previously unrecognized role in regulating CLDN1 and OCLN localization on the cell surface and is an important co-factor in HCV entry. Another identified host factor is SMAD6, an inhibitory SMAD (I-SMAD) of the TGF- signaling pathway. We further investigated the role and mechanism of SMAD6 in modulating HCV infection. We employed various HCV in vitro models to explore the mechanism of action of I-SMADs on HCV infection and characterized cellular signaling pathways regulated by I-SMADs in both hepatoma cell lines and primary human hepatocytes. The expression of I-SMADs and other genes was also studied in HCV-infected hepatocytes and human livers from chronic hepatitis C patients. SMAD6 and its I-SMAD partner SMAD7 enhance HCV entry, predominantly at the viral attachment step, through transcriptionally up-regulation of heparan sulfate proteoglycan (HSPG) expression on cell surface. SMAD6 is also involved in the expression of multiple lipoprotein and cholesterol uptake receptors, including LDLR and SR-BI in hepatocytes. I-SMADs and HSPG levels are elevated in HCV-infected hepatoma cells, primary human hepatocytes and liver biopsies from HCV infected patients. I-SMADs enhance HCV attachment and entry via up-regulation of cellular entry factors including HSPGs, LDLR and SR-BI. HCV infection in turn enhances expression of I-SMADs and downstream signaling pathways as a strategy to facilitate viral propagation. Our study thus reveals a novel I-SMAD-mediated pathway that facilitates HCV entry by transcriptional regulation of hepatic HSPG contents and cholesterol uptake. Cellular miRNAs have been shown to regulate hepatitis C virus (HCV) replication, yet a systematic interrogation of the entire repertoire of miRNAs impacting HCV life cycle is lacking. Using the same screening technology, we performed an unbiased strategy to identify cellular microRNAs associated with HCV infection and functionally interrogate these miRNAs with our previous HCV small interference RNA (siRNA) screen database to derive an extensive cellular/viral regulatory network in productive HCV infection. Through genome-wide miRNA mimic and hairpin inhibitor phenotypic screens, and miRNAmRNA transcriptomics analyses, we identified three proviral and nine antiviral miRNAs that interact with HCV in a physiologically relevant manner. These miRNAs were then functionally linked to particular steps of HCV life cycle and relevant viral host dependencies, thereby revealing extensive cellular miRNAmRNA regulatory networks associated with HCV infection and propagation. Further mechanistic studies demonstrated that miR-25, let-7 and miR-130 families repress essential HCV co-factors, thus restricting viral infection at multiple stages. HCV meanwhile subverts the antiviral actions of these miRNAs by downregulating their expression in cell culture and HCV-infected human livers. This comprehensive HCVmiRNA interaction map provides fundamental insights into HCV-mediated pathogenesis and unveils molecular pathways linking RNA biology to viral infections.